PROJECT SUMMARY We have made great advances in our ability to treat cancer. However, we still lack the ability to determine which patients will benefit the most from treatment. In head and neck squamous cell carcinoma (HNSCC), the decision to treat with radiation and chemotherapy is based on anatomical evaluations of tumor stage and progression. In addition, treatment response is evaluated several weeks post-completion of therapy. An early determination of treatment resistance would greatly alleviate the pain and suffering for patients with treatment-resistant tumors who would otherwise undergo several weeks of ineffective therapy. Three research teams with complementary expertise in diffuse reflectance spectroscopy, Raman spectroscopy, and radiation biology have joined forces to develop a non-invasive, quantitative tool that can reveal key metabolic, functional and molecular changes in response to radiation and chemotherapy in HNSCC. Specifically, this multi-modal optical sensing approach affords simultaneous, real-time determination of tumor oxygenation and metabolism that play key complementary roles in shaping treatment resistance. The integration of diffuse reflectance and Raman spectroscopic modalities is motivated by our preliminary data acquired from radiation-resistant and sensitive tumor xenografts that shows significantly higher reoxygenation and elevated lipid and glycogen content in the radiation-resistant tumors. This application seeks to significantly advance these preliminary findings for comprehensive characterization of radiation and chemotherapeutic responses. Aim 1 is focused on pre-clinical studies using diffuse reflectance spectroscopy to investigate short-term and long-term reoxygenation kinetics of chemo and radiation-resistant and sensitive tumors in response to clinical treatment regimens and, in the process, identify time-dependent thresholds that can reliably predict radiation resistance. In Aim 2, we will investigate the dynamic changes in tumor metabolism after radiation and chemotherapy using Raman spectroscopy and correlate these findings with metabolomics to map the key spectral features to molecular determinants. Finally, in Aim 3, we seek to translate the consolidated panel of spectroscopic markers through a pilot clinical study in patients with HNSCC who are scheduled for chemoradiation therapy. To determine the initial sensitivity of our approach in identifying treatment-resistant tumors within the first half of the therapeutic regimen, we will develop and employ a multimodal inverse spatially offset Raman and diffuse reflectance spectroscopy probe that is compatible with a standard clinical laryngoscope. Successful completion of these aims will delineate functional and molecular changes associated with radiation and chemoresistance at unprecedented time scales. This knowledge of radiobiological changes occurring immediately after therapy will not only aid in differentiating treatment responders and non-responders but also identify additional time points at which meaningful changes to therapy could improve treatment response rates.